trackingthefearmemoryengram:discretepopulations of...

Research Paper

Tracking the fear memory engram: discrete populationsof neurons within amygdala, hypothalamus, and lateralseptum are specifically activated by auditory fearconditioningChristopher W. Butler,1 Yvette M. Wilson,1 Jenny M. Gunnersen, and Mark Murphy

Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, Victoria 3010, Australia

Memory formation is thought to occur via enhanced synaptic connectivity between populations of neurons in the brain.However, it has been difficult to localize and identify the neurons that are directly involved in the formation of any specificmemory. We have previously used fos-tau-lacZ (FTL) transgenic mice to identify discrete populations of neurons in amygdalaand hypothalamus, which were specifically activated by fear conditioning to a context. Here we have examined neuronalactivation due to fear conditioning to a more specific auditory cue. Discrete populations of learning-specific neuronswere identified in only a small number of locations in the brain, including those previously found to be activated in amyg-dala and hypothalamus by context fear conditioning. These populations, each containing only a relatively small number ofneurons, may be directly involved in fear learning and memory.

Pavlovian or classical conditioning involves the presentation of aneutral stimulus, the conditioned stimulus (CS), paired with a bio-logically significant stimulus, the unconditioned stimulus (US),such as pain or food. Once learning has occurred, presentationof the CS alone is able to elicit a response, and this learned reac-tion to the previously neutral CS is known as the conditionedresponse. Fear conditioning is a robust type of classical condition-ing, which uses an aversive US such as a footshock. It is one of themost well characterized and extensively studied models of learn-ing and memory (Paré 2002; Sah et al. 2003; Maren and Quirk2004; Fanselow and Poulos 2005; Kim and Jung 2006; Ehrlichet al. 2009; Johansen et al. 2011).

A number of major brain regions have been identified whichare required for fear conditioning (LeDoux 2000). However, spe-cific neurons within these regions and throughout the brainthat are directly involved in fear conditioning have not been iden-tified. The identification of these neurons is required to elucidatethe memory trace or engram. Thus, the identification of theseneurons is required to determine the neuronal changes that are in-volved with and encode the fear memory, to define the precise cir-cuits within the brain that are involved in fear learning andmemory, and to determine how the neuronal changes affectthose neuronal circuits to establish the memory. One methodfor identifying neurons activated by a certain task is to use theimmediate-early gene c-fos as a marker of neuronal activation. Anumber of studies have examined c-fos expression following fearlearning (Smith et al. 1992; Beck and Fibiger 1995; Radulovicet al. 1998), implicating a number of different brain regions in-cluding parietal cortex, hippocampus, and amygdala. However,these studies did not determine if the c-fos expression was specif-ically associated with fear learning or whether it was related to dif-ferent sensory stimuli the animals received (Radulovic et al. 1998).

More recent studies have utilized c-fos-regulated expressionin transgenic mice to identify neuronal populations that are acti-

vated by exposure to specific contexts during fear conditioning(Reijmers et al. 2007; Garner et al. 2012; Liu et al. 2012; Ramirezet al. 2013). Subsequent specific inhibition or activation of theseneurons directly demonstrated that these c-fos-activated neuronswere involved in the memory of specific contexts (Garner et al.2012; Liu et al. 2012; Ramirez et al. 2013). These experimentsnot only show that different subpopulations of hippocampal neu-rons are involved in memory of different contexts, but also vali-date the approach of using fos-regulated expression as a markerfor neurons involved in learning and memory.

Our previous development of the transgenic fos-tau-lacZ(FTL) mouse (Wilson et al. 2002; Murphy et al. 2007) has emergedas an excellent tool for visualizing functionally activated earlygene expression in brain subnuclei associated with learning andmemory. Previously, we trained FTL mice using context fear con-ditioning and identified a number of discrete, anatomicallydefined populations of neurons within amygdala and hypothala-mus which were specifically activated by learning (Wilson andMurphy 2009; Trogrlic et al. 2011). While context fear condition-ing is a very robust form of fear conditioning, the context CS iscomplex and relatively undefined, and could include differentcombinations of visual, olfactory, auditory, and tactile stimuli.It is thus difficult to determine the relative importance of thesedifferent stimuli in the fear conditioning process, and the popula-tion of activated neurons may be less specific than if a more pre-cise CS is used.

Auditory fear conditioning, using a specific tone as CS, is amore studied and better characterized form of fear learning. Wehypothesized that the populations of neurons specifically activat-ed by this form of fear conditioning would represent a more tight-ly defined subset of the populations activated by context fearconditioning and we have trained FTL mice to determine whether

1These authors contributed equally to this work.Corresponding author: [email protected]

# 2015 Butler et al. This article is distributed exclusively by Cold SpringHarbor Laboratory Press for the first 12 months after the full-issue publicationdate (see http://learnmem.cshlp.org/site/misc/terms.xhtml). After 12months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.037663.114.

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this is the case. In addition, we have undertaken a brain-widescreen of FTL activation, with the exclusion of cortical regions.To maximize the specificity of learning, we modified the tradi-tional auditory fear-conditioning paradigm so that associationof the footshock to the context of the training chamber is mini-mized (Nithianantharajah and Murphy 2008). We find a limitednumber of discrete, anatomically defined populations of neuronsthat are activated by auditory-specific fear conditioning, withsome overlap in those regions activated by context fear learning.

Results

Behavioral resultsTo identify neurons specifically activated by auditory fear learn-ing, we used a protocol in which animals were trained to specifi-cally fear an auditory stimulus, with no fear response to othersensory stimuli that they were exposed to during training (seeNithianantharajah and Murphy 2008). Mice were habituated tothe context of the fear-conditioning chamber for 3 min eachday for 3 wk prior to training, so that the association of fear to con-textual cues would be minimal. Mice were also exposed to an en-riched environment during this period as we showed previouslythat auditory fear learning outcomes are improved by this enrich-ment protocol (Nithianantharajah and Murphy 2008). Followingthe period of habituation and environmental enrichment, thePaired group of mice were conditioned to fear an auditory toneby placing them in the conditioning chamber and playing thetone for 30 sec, co-terminating with a footshock (Fig. 1).

A relatively low shock level (0.2 mA) was used. In previous ex-periments in context fear conditioning, we used low shock levelsbecause the fear-conditioned mice showed clear, robust freezingwhereas control mice showed only very low levels of freezing(Wilson and Murphy 2009; Trogrlic et al. 2011). At higher shocklevels, shock-control mice still showed significant levels of freez-ing (Nithianantharajah and Murphy 2008) indicating some learn-ing had occurred.

We useda numberof control groupsof mice that enabledus todistinguish between FTL activation due to learning and to non-learning experiences (Fig. 1). To control for experience of context,the Context group was exposed only to the conditioning chamberduring training. Similarly, the Tone group was exposed to both thecontext of the chamber as well as the auditory tone, but with no

footshock. The Unpaired group was exposed to a temporally un-paired tone and shock, thereby providing a control condition inwhich animals have been exposed to the same set of sensory stim-uli as Paired mice, but have not encoded a fear memory. Inaddition, a Recall group of mice was included, enabling us todetermine if neuronal populations that were activated by learningwere also activated by the recall and expression of fear. Recall micewere first trained using the paired protocol, and then returned totheir home cages for 72 h (giving enough time for FTL transgeneexpression to return to basal levels [Wilson and Murphy 2009]).

To determine if the mice had acquired a fear response, eachmouse was reexposed to the fear conditioning chamber and audi-tory tone 4 h after training (excluding Recall mice, as describedabove). The first measure of fear expression was the percent oftime the mice spent freezing (see Fig. 2A,B). Context, Tone, andUnpaired mice did not freeze more than base levels to the fear con-ditioning chamber during testing, either in the presence or ab-sence of the auditory tone, indicating they had not learned tofear either the context of the conditioning chamber or the audito-ry tone (Fig. 2A). Only Paired mice froze significantly morethan base levels in the presence of the auditory tone (t ¼ 8.627,P , 0.001). Thus, only Paired mice learned to fear the auditorytone, and this fear memory was specific to the tone and not tothe chamber context (Tone effect F(1,32) ¼ 15.95, P ¼ 0.0004,treatment effect F(3,32) ¼ 20.80, P , 0.0001, interaction F(3,32) ¼14.32, P , 0.0001). The time each mouse spent moving was alsorecorded as a second measure of fear (Fig. 2C,D). Only Pairedmice moved significantly less in the presence of the tone (Fig.2C; t ¼ 7.792, P , 0.001), again indicating that only the Pairedgroup of mice were conditioned to fear the tone (Tone effectF(1,32) ¼ 10.53, P ¼ 0.0028, treatment effect F(3,32) ¼ 5.85, P ,0.0026, interaction F(3,32) ¼ 12.91, P , 0.0001).

Recall mice were reexposed to the context and tone after 48 hin their home cage, and froze significantly more when presentedwith the auditory tone than without tone presentation (Fig. 2B;t(8) ¼ 4.622, P ¼ 0.0017). Moving was also significantly reducedin Recall mice in the presence of the tone (Fig. 2B; t8 ¼ 4.487,P ¼ 0.002; Fig. 2D).

Analysis of learning-induced FTL expressionMice were terminally anesthetized immediately after fear testingand brain sections from each treatment group were qualitatively

examined for regions showing differ-ences in FTL expression between theauditory fear learning group and thenonlearning control groups. Regions ofinterest were described anatomically,and counts of FTL+ neurons were under-taken in defined areas as described below.

Learning-induced activation in theamygdalaWe previously described a population ofneurons specifically activated by contextfear learning in the ventrolateral sub-division of the lateral amygdala (LAvl)(Wilson and Murphy 2009; Trogrlicet al. 2011). Interestingly, a similar distri-bution of FTL+ neurons was identified inthe current experiment following audito-ry fear conditioning (Fig. 3). FTL+ neu-rons were identified in Paired mousebrains, along the lateral border of LAvl,between bregma 21.7 and 22.0 mm.

Figure 1. Training and testing paradigm for each group of mice. During training Paired mice wereexposed to a paired presentation of the auditory tone and footshock. Four hours after training Pairedmice were tested for auditory fear memory by exposing them to the training chamber and the auditorytone. Unpaired mice were exposed to a temporally unpaired tone and shock during training, and tested4 h later. Tone mice were exposed to the training chamber and the auditory tone, and tested 4 h later.Context mice were exposed to only the context of the training chamber, and tested 4 h later. Paired,Unpaired, Tone, and Context mice were anesthetized immediately following testing, and perfused.Recall mice were trained as Paired mice, but returned to their home cages for 72 h. Recall mice werethen tested, anesthetized, and perfused 4 h later. Home Cage mice had no training or testing.

Neurons specifically activated in auditory fear learning

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FTL+ neurons were counted within a 100-mm wide rectanglealigned with the lateral border of the LAvl (see Fig. 3A,C), extend-ing the length of the LA in each brain section. There was a four- tosixfold increase in FTL+ neurons in Paired brains compared withUnpaired and Recall, respectively (F(5,43) ¼ 51.93, P , 0.0001;Bonferroni’s multiple comparison test: Paired versus Unpaired:P , 0.001; Paired versus Recall: P , 0.001). These FTL activatedneurons are a small, discrete population of learning-specific neu-rons along the border of the LA. We estimate that they represent5% of neurons in the border region of the LAvl (as described inMaterials and Methods).

Very few FTL+ neurons were identified in this region inHome Cage. Further, exposure to context did not result in any in-crease in FTL activation (P . 0.05), which may have been due toprior treatment of the mice to EE and habituation to the condi-tioning chamber. There was also no significant increase in thenumber of FTL+ neurons following exposure to context in anyof the other regions described below (P . 0.05).

Additionally, there were no effects of auditory fear condition-ing on FTL expression in the other divisions of the lateral amygda-la, the dorsal (LAd) and ventromedial (LAvm) nuclei (Fig. 4; LAdF(5,39) ¼ 1.099, P ¼ 0.3766; LAvm F(5,39) ¼ 1.016, P ¼ 0.4217).Similarly, we did not find a learning-specific increase in FTL+ neu-rons in the basolateral amygdala (Fig. 4; F(5,39) ¼ 0.5598, P ¼0.730).

Intense learning-specific FTL expression was also observedwithin the posterodorsal nucleus of the medial amygdala(MeApd; Fig. 5). The learning-specific expression was most prom-inent in the dorsal region of the MeApd adjacent to the optic tractand between bregma 21.06 mm and bregma 21.58 mm. FTL+

neurons were counted in each treatment group in the MeApd,within a rectangular region 250mm wide positioned on the border

of the optic tract (Fig. 5A,B), extending the length of the optictract in each brain section. In this region, there was at least a five-fold increase in the number of FTL+ neurons in the Paired group(F(5,43) ¼ 14.69, P , 0.0001; Bonferroni’s multiple comparisontest comparing Paired versus Unpaired: P , 0.001; Paired versusRecall: P , 0.001). Additionally, and as described above for theLAvl, the neurons showing FTL activation in the Paired mice area small population, representing �1.2% of neurons within this re-gion of the MeApd.

The amygdalostriatal transition area (AStr) also showedlearning-specific expression (Fig. 5A,B). These FTL+ neuronswere observed between bregma 21.06 and 21.94 mm in theAStr region, with four times as many FTL+neurons in Paired brainscompared with Unpaired brains. A small increase in FTL+ neuronswas also observed in Unpaired brains compared with Context,Tone, and Homecage. Therewas a significant increase in FTL+neu-rons in the Paired AStr (F(5,43) ¼ 23.71, p , 0.0001; Bonferroni’smultiple comparison test comparing Paired versus Unpaired: P ,0.001; Paired versus Recall: P , 0.001). The population of FTL+

neurons in Paired mice represented �9% of neurons in the AStr.Learning-specific FTL+ neurons were observed within the

capsular region of the central amygdala (CeC, Fig. 6). FTL+ neu-rons were found along the border of the CeC adjacent to the baso-lateral amygdala, extending rostro-caudally from bregma 20.80–0.95 mm (Fig. 6). In rostral regions of the CeC, these learning-specific neurons were relatively ventral but their location becamemore dorsal through successive caudal regions of CeC (Fig. 6A1–4). In this region of the CeC, there was a fourfold increase in FTL+

neurons in Paired compared with Unpaired brains (Fig. 6C). Aswith the AStr population, there was also a small increase inFTL+ neurons in Unpaired brains when compared with theContext, Tone, and Home Cage brains. There was a significant in-crease in FTL+ neurons in the Paired CeC (F(5,43) ¼ 21.82, P ,0.0001; Bonferroni’s multiple comparison test comparing Pairedversus Unpaired: P , 0.001; Paired versus Recall: P , 0.001). Thepopulation of FTL+ neurons in Paired mice represented �3.6%of neurons in this region of the CeC.

These results suggest that each of the amygdala populationsof neurons described above may be specifically involved in audito-ry fear conditioning, and the lack of FTL expression in recallbrains suggests that FTL activation was not due to fear memory re-call or expression of fear.

Learning-induced activation in the hypothalamusWe previously identified a population of neurons within the hy-pothalamus that was specifically activated following contextfear conditioning (Trogrlic et al. 2011). We found a similar popu-lation of neurons activated by auditory fear conditioning in therostral region of the ventromedial hypothalamus (VMH; bregma21.04–1.44 mm; Fig. 7). There was a ninefold increase in FTL+

neurons in this region in Paired mice compared with Unpairedmice (Fig. 7K). Very few FTL+ neurons were counted in theHome Cage, Context, and Tone control groups. The increase inFTL+ neurons in Paired brains was significant (F(5,43) ¼ 36.42,P , 0.0001, Bonferroni’s multiple comparison test comparingPaired versus Unpaired: P , 0.001; Paired versus Recall: P ,0.001). The population of FTL+ neurons in Paired mice represent-ed �6% of neurons in this region of the VMH.

An additional population of learning-specific FTL expressionwas identified in a more caudal region within the VMH. Theseneurons correspond to the ventrolateral subdivision of the VMH(VMHvl; Franklin and Paxinos 2008), between bregma 21.7 mmand bregma 21.8 mm (Fig. 7L). There was a 13-fold increase inFTL+ neurons in this region of the VMHvl in Paired comparedwith Unpaired mice, with very few FTL+ neurons observed in

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Figure 2. Fear expression in mice during testing. (A) Four hours aftertraining, only Paired mice froze in response to the auditory tone, andlevels of freezing to the training chamber alone was not significant. (B)Recall mice froze significantly more when exposed to the auditory tone72 h after training. (C) Four hours after training, only Paired mice showa reduction in movement in response to the auditory tone, and therewas no significant decrease in movement without tone. (D) Recall miceshowed significantly less movement when exposed to the auditory tone72 h after training. Numbers are expressed as percent time freezing (Aand B) or moving (C and D) + SEM. (∗∗) P , 0.01 and (∗∗∗) P , 0.001.





the other control mice. There was a sig-nificant increase in FTL+ neurons withinthe Paired VMHvl (F(5,42) ¼ 9.94, P ,0.0001, Bonferroni’s multiple compari-son test comparing Paired versus Un-paired: P , 0.001; Paired versus Recall:P , 0.001). The population of FTL+ neu-rons in Paired mice represented �8.7% ofneurons in this region of VMHvl.

These results indicate that two spe-cific populations of neurons in theVMH are activated by auditory fear con-ditioning, and the low numbers of FTL+

neurons in the VMH of recall mice sug-gests that FTL activation is not associatedwith the expression of fear.

Learning-induced activationin the lateral septumA discrete region within the lateral sep-tum was also found to show learning-associated activation following audi-tory fear conditioning. At approximatelybregma 0.30 mm, a region containedwithin a 400 × 600-mm oval either sideof the midline within the intermediatesubdivision of the Lateral septum (LSI)was found to be strongly FTL+ in Pairedbrains (Fig. 8). Counts of FTL+ neuronswithin this region of LSI are shown inFigure 8F. There were significant num-bers of FTL+ neurons in all conditions,but Paired mice contained at least 60%more FTL+ neurons compared with allother groups. There was a significant ef-fect of treatment on number of FTL-la-beled neurons (F(5,42) ¼ 5.7, P ¼ 0.0004,Bonferroni’s multiple comparison testcomparing Paired versus Unpaired: P ,0.05; Paired versus Recall: P , 0.05). Thepopulation of FTL+ neurons in Pairedmice represented �4.4% of neurons inthis region of LSI.

Regions with no learning-inducedactivationThere were some other areas of thebrain that may have had learning-relatedFTL expression when screened quali-tatively. These were Ventral TegmentalArea, dorsal lateral Geniculate Nucleus,Pregeniculate Nucleus, and dorso-lateralPeriaqueductal Gray. However, uponquantitative analysis of these regions,none were found to have significantlymore FTL+ neurons when comparedwith nonlearning controls (ventral teg-mental area; F(5,40) ¼ 4.659, Paired versusUnpaired, P . 0.05; dorsal lateral Genic-ulate Nucleus, F(5,40) ¼ 1.785, Paired ver-sus Unpaired P . 0.05; PregeniculateNucleus, F(5,40) ¼ 4.530, Paired versusUnpaired, P . 0.05; dorso-lateral Peria-queductal Gray, F(5,42) ¼ 1.581, Paired

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Figure 3. Learning-specific FTL+ neurons in ventrolateral Lateral Amygdala (LAvl) following auditoryfear conditioning. (A) Bright-field photograph of amygdala at bregma 21.7 mm showing FTL+ neuronsin the LAvl in a Paired trained mouse. The solid box encloses the area shown in high power in B. Countedregion is encompassed by dotted line. (B) High-power view of FTL+ neurons in Paired LAvl. (C)Bright-field photograph of Unpaired trained mouse at bregma 21.7 mm. The solid box encloses thearea shown in high power in D. Counted region is encompassed by dotted line. (D) High-powerview of Unpaired LAvl boxed in C. (E) Plate from Mouse Brain Atlas (Franklin and Paxinos 2008) high-lighting the region shown in A and C. (F) FTL+ neuron counts of LAvl region in each group of trainedmice, shown as mean+SEM. Significantly more FTL+ neurons were present in Paired mouse LAvl com-pared with control groups. (∗∗∗) P , 0.001. Scale bar, 250 mm (A,C), 100 mm (B,D).





versus Unpaired, P . 0.05). No other brain regions were detectedwhich showed any learning-specific activation.

DiscussionIn this study, we aimed to identify discrete populations of neuronsin the brain that were specifically activated by auditory fear learn-ing. We trained FTL mice using a model of fear conditioning inwhich the mice learn to specifically fear an auditory cue with littleor no fear of any other contextual stimuli. The use of FTL mice al-lows a relatively straightforward method for imaging the brain.The advantage over Fos immunohistochemistry or c-fos in situ hy-bridization results from two principal characteristics of the FTL

transgene. First, expression of the transgene is targeted to cell bod-ies and their processes, and second, transgene activation can bedetected either bybgal histochemistry or immunohistochemistry.This allows imaging from the macroscopic level (e.g., whole re-gions of the brain) to a high-resolution level (e.g., a single neuro-nal process). In comparison, c-fos expression is restricted to cellnuclei, and thus imaging using conventional c-fos detection tech-niques does not allow either simple macroscopic imaging or high-resolution imaging and analysis of cell bodies and their processes.In this study, FTL imaging using histochemistry permitted the ef-ficient screening of sections across the brain and the detection ofspecific patterns of neuronal activation, such as clusters of similar-ly shaped FTL+ neurons.

We identified a very limited number of regions in the brainthat were specifically activated by auditory fear learning, withlearning-specific populations found in the amygdala, hypothala-mus and lateral septum. We previously showed that some of thesepopulations in amygdala and hypothalamus were activated fol-lowing the encoding of context fear memories (Trogrlic et al.2011), while the current findings suggest that either similar oroverlapping populations of neurons are directly involved in theprocess of auditory fear learning.

A number of control groups of animals were used to omitthose neurons that were activated due to nonlearning events orstimuli. Mice in the main control group, Unpaired, were exposedto the same sensory stimuli as the Paired group, but did not learn.However, unlike the Paired mice, Unpaired mice do not expressfear. We utilized another important control, the Recall group,which enabled us to determine if neuronal populationsthat showed FTL activation following learning also showed FTLactivation following the recall of fear. In our previous studieswith context fear conditioning, we found none of the neuronalpopulations that showed FTL activation following learning alsoshowed FTL activation following recall (Trogrlic et al. 2011).This finding demonstrated that FTL expression in these popula-tions was not due to fear expression. Comparison of the learninggroup with each of these control groups enabled the identificationof neuronal ensembles that were specifically activated by thelearning of auditory fear conditioning.

It is important to note that lack of FTL (and c-fos) expressionin neurons during recall does not preclude involvement of thoseneurons in the recall process. These neurons may well fire duringrecall, but with no subsequent c-fos expression. Induction of c-fosnot only requires depolarization and increases in firing rate, but isalso associated with strong activation of neurotransmitter recep-tors and substantial changes in intracellular Ca2+ (Cirelli andTononi 2000; Kovacs 2008). Thus, c-fos activation is consideredto be indicative of strong activation of cell functioning during pe-riods of plasticity or high rates of metabolic activity, and not sim-ply a marker of neuronal firing (Cirelli and Tononi 2000; Kovacs2008). The learning-specific FTL expression occurring followingfear conditioning may thus coincide with plasticity underlyingmemory formation. We have previously identified different pop-ulations of neurons in hypothalamus and other brain regionsshowing FTL activation after recall of context fear conditioning(Ali et al. 2012).

A modified auditory fear conditioning paradigm was used tominimize the association of the footshock to more general andless defined contextual cues. This involved extensive habituationto the shock chamber and concurrent exposure to an enriched en-vironment (Nithianantharajah and Murphy 2008). This model ofauditory fear conditioning thus enables us to obtain a more specif-ic neural correlate of conditioned fear than that attained usingcontext fear conditioning. Overall levels of FTL expression inthe current study appeared less than those observed previously(Trogrlic et al. 2011), which may be due to the pretraining

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Figure 4. FTL+ neuron numbers in other basolateral amygdala subdivi-sions. (A) Total numbers of FTL+ neurons in the basolateral amygdala(BLA) in each group of trained mice. (B) Total numbers of FTL+ neuronsin the dorsal lateral amygdala (LAd) in each group of trained mice. (C)Total numbers of FTL+ neurons in the medial lateral amygdala (LAm) ineach group of trained mice. Numbers are expressed as mean+SEM.No significant difference in FTL+ neuron number was observed betweenany of the groups in any of these amygdala regions.





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Figure 5. Learning-specific FTL expression in the Medial Amygdala and Amygdalostriatal Transition Region following auditory fear conditioning. (A)Bright-field photograph of FTL+ neurons at bregma 21.22 mm in a Paired mouse. Solid boxes enclose areas that are shown in high power in B andC. Regions that were counted are enclosed by dotted lines. (B) High-power view of FTL+ neurons in a Paired mouse AStr. (C) High-power view ofFTL+ neurons in MeA from a Paired mouse. (D) Bright-field photograph of section taken at bregma 21.22 mm in an Unpaired trained mouse. Solidboxes encompass areas that are shown in high power in E and F. Regions that were counted are encompassed by dotted lines. (E) High-power viewof AStr from an Unpaired mouse. (F) High-power view of Unpaired MeA. (G) Plate from Mouse Brain Atlas (Franklin and Paxinos 2008) highlightingthe regions shown in A and D. (H) FTL+ neuron counts of MeA region in each group of mice, shown as mean+SEM. There were significantly moreFTL+ neurons in Paired mouse MeA compared with controls. (I) FTL+ neuron counts of AStr region of each group of mice. There were significantlymore FTL+ neurons in Paired AStr compared with controls. (∗∗∗) P , 0.001. Scale bar, 500 mm (A, D), 50 mm (B, C, E, F).





habituation and/or environmental enrichment. Habituation andhandling of mice reduces c-fos expression in many areas of thebrain (Papa et al. 1993; Asanuma and Ogawa 1994; Ryabininet al. 1999).

Environmental enrichment results in a series of behavioraland brain changes in the mature animal (Diamond 2001;Nithianantharajah and Hannan 2006). These include improve-ments in learning and memory, decreased stress, increased

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Figure 6. Learning-specific FTL+ expression in CeC following auditory fear conditioning. (A1–4) Images of a series of sections from a Paired brainshowing FTL+ neurons in CeC from bregma 20.8–1.0 mm. Region counted is shown encompassed by dotted line. (B1–4) Comparative series ofimages from an Unpaired brain. (C) FTL+ neuron counts of this region in each group of trained mice, shown as mean+SEM. There were significantlymore FTL+ neurons in Paired mouse brains compared with controls. (D) Plate from Mouse Brain Atlas (Franklin and Paxinos 2008) highlighting theregion shown in A1–4 and B1–4. (E) High-power view of Paired FTL+ neurons from A3. (∗∗∗) P , 0.001. Scale bar, 100 mm (A1–4, B1–4), 50 mm (E).





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Figure 7. Learning-specific FTL+ neurons in the VMH and VMHvl following auditory fear conditioning. (A) Bright-field photograph of hypothalamicregion at bregma 21.2 mm showing FTL+ neurons in the VMH in a Paired mouse. The solid box encloses the area shown in high power in C.Neurons were counted in region encompassed by dotted line. (B) Comparative photograph of Unpaired mouse. (C) High-power view of FTL+

neurons in Paired VMH. (D) High-power view of Unpaired VMH boxed in B. (E) Plate from Mouse Brain Atlas (Franklin and Paxinos 2008) highlightingthe region shown in A and B. (F) Bright-field photograph of hypothalamic region at bregma 21.8 mm from a Paired mouse. The solid box enclosesthe area shown in high power in H. Neurons were counted in region encompassed by dotted line. (G) Comparative image from an Unpaired mouse.(H) High-power view of FTL+ neurons in Paired VMHvl. (I) High-power view of VMHvl from an Unpaired mouse boxed in G. (J) Plate from MouseBrain Atlas (Franklin and Paxinos 2008) containing the region shown in F and G. (K) FTL+ neuron counts of rostral VMH region in each group oftrained mice. Significantly more FTL+ neurons were present in rostral VMH from Paired mice compared with controls. (L) FTL+ neuron counts ofcaudal VMHvl region in each group of trained mice. There were significantly more FTL+ neurons in Paired mouse VMHvl compared with controls.Numbers are expressed as mean+SEM. (∗∗∗) P , 0.001. Scale bar, 250 mm (A, B, F, G), 50 mm (C, D, H, I).





cortical thickness and neuronal size, changes in synapse size andnumber, and increased neurogenesis (Kempermann et al. 1997).Our studies suggest environmental enrichment induces relativelywidespread changes in the brain (Nithianantharajah et al. 2004),including effects on FTL expression at the start of enrichment (Aliet al. 2009). Given that environmental enrichment results in de-creased stress levels and stress has long been known to increasec-fos expression (Pezzone et al. 1992; Smith et al. 1992), environ-mental enrichment over a period of weeks may result in generallydecreased c-fos expression in the brain. Combined with improvedlearning and memory, it would thus be expected that environ-mental enrichment would improve the specificity of the neuralcorrelate of conditioned fear.

C-fos is a widely expressed gene,however its lack of expression in a brainregion does not necessarily preclude neu-ronal activation (Labiner et al. 1993).Thus, we cannot detect neurons that donot utilize c-fos during learning, suchas many types of inhibitory neurons.Further, it could not be determined ifthere was learning-specific expression insome areas of the brain with high levelsof baseline FTL expression, such as cor-tex. Finally, there are some areas of thebrain that most likely contribute to audi-tory fear learning, but are not specificallyinvolved and activated in the formationof an association between the auditorytone and the footshock. For example,in our analysis of context fear condition-ing, we did not detect learning-specificactivation in hippocampus; this struc-ture was activated by both context ex-posure and fear learning (M Murphyand Y Wilson, unpubl.). This is consis-tent with the requirement of the hip-pocampus in context fear memorythrough its coding for the context(Garner et al. 2012; Liu et al. 2012).

Learning-inducedneuronal activation in theamygdalaThe population of FTL+ neurons seenwithin LAvl is very similar in distributionto that seen following context fear con-ditioning (Wilson and Murphy 2009;Trogrlic et al. 2011). As previously dis-cussed (see Trogrlic et al. 2011), the LAhas been implicated as a site of CS–US as-sociation due to the convergence of CSand US sensory signals (Rodrigues et al.2004; Kim and Jung 2006; Ehrlich et al.2009). Thus, our hypothesis that thec-fos-related expression within the LAvlneurons following learning may be relat-ed to the formation of the CS–US associ-ation (Wilson and Murphy 2009; Trogrlicet al. 2011) is further supported by theresults of the current study. Whetherthe same neurons are activated by bothcontext and auditory fear conditioningrequires further examination. The acti-vated neurons we identified still only

represent a very small percentage of the total neurons withinthis region of the LA and thus it may be that the auditory andcontext-specific neurons may be separate or overlapping.

It is also known that phosphorylation of ERK/MAP kinase inthe LA is required for auditory fear conditioning in the rat (Schafeet al. 2000). Auditory fear conditioning results in activation ofMAP kinase throughout the LA, in particular along the lateral bor-der of the LA, in LAd and LAvl (Schafe et al. 2000; Bergstrom andJohnson 2014). Given that MAP kinase activation partly regulatesc-fos expression (Adams and Sweatt 2002), it may be that there isoverlap in the FTL+ and MAP kinase activated populations inthe LAvl. In any case, these data provide further evidence that dis-crete, and possibly overlapping, populations of neurons along the

A

C D

E F

B

Figure 8. Learning-specific FTL+ neurons in lateral septum (LSI) following auditory fear conditioning.(A,C) Bright-field images of LSI at bregma 0.3 mm from a Paired and Unpaired mouse, respectively. Thesolid box encloses the area shown in high power in B. Counted region is encompassed by dotted line.(B,D) High-power views of FTL+ neurons in Paired and Unpaired LSI, respectively. (E) Plate from MouseBrain Atlas (Franklin and Paxinos 2008) highlighting the region shown in A and C. (F) FTL+ neuroncounts of LSI region in each group of trained mice, shown as mean+SEM. Significantly moreFTL+ neurons were present in Paired mouse LSI region compared with controls. (∗) P , 0.05, (∗∗)P , 0.01, (∗∗∗) P , 0.001. Scale bar, 500 mm (A, C), 100 mm (B, D).





lateral border of the LA are activated and involved in auditory- andcontext fear learning, respectively.

We found no increase in numbers of FTL+ neurons followingrecall of auditory or context fear memory (Trogrlic et al. 2011)compared with basal levels, indicating that these neurons donot express c-fos following fear memory recall. One interpretationof this finding is that these neurons undergo plasticity followingfear learning associated with the formation of the primary fearmemory, but there is no further plasticity following fear memoryrecall. If this were the case, learning-induced plasticity may havealtered the firing properties of these neurons and contributed tothe conditioned behavior of the mice, but no further changes oc-curred following recall.

Medial amygdalaThe MeA was another site of learning-specific FTL activation fol-lowing both context- and auditory-fear conditioning (Trogrlicet al. 2011). The learning-specific expression was located in dorsalregions of MeApd. A series of prior studies have examined c-fosexpression in MeA using different fear learning models, butnone have determined if the c-fos expression was specifically as-sociated with learning (Pezzone et al. 1992; Campeau et al.1997; Milanovic et al. 1998; Radulovic et al. 1998; Rosen et al.1998; Trogrlic et al. 2011).

A number of predator odor-induced fear experiments impli-cate the MeA in fear learning and memory. Predator odor is in-nately aversive for rodents and can therefore be used as a US infear conditioning. Ibotenic acid lesions to the rat MeA reducefreezing in response to cat-odor exposure (Li et al. 2004), and tem-porary inhibition of the rat MeA with GABA agonist, muscimol,completely blocks the expression of fear to fox odor (Müller andFendt 2006). Thus, the MeA is required for the processing of pred-ator odor-induced fear. In addition, lesions of MeA after predatorodor exposure impaired context fear conditioning (Takahashiet al. 2007). These findings suggest that the MeA is requiredfor fear memory of the context in which a predator odor wasexperienced.

Because of this experimental evidence linking MeA with ol-factory stimuli, we previously hypothesized that the activationin MeA following context fear conditioning may be related to un-defined olfactory cues within the conditioning chamber (Trogrlicet al. 2011). However, this is not consistent with the current study,given we have specifically conditioned mice to an auditory stim-uli. Whereas lesioning or inhibition of MeA does not impair audi-tory or context fear conditioning (Nader et al. 2001; Walker et al.2005), another form of fear memory, fear potentiated startle, is af-fected (Walker et al. 2005). Recent studies also indicate that MeAmediates aversive Pavlovian instrumental transfer (McCue et al.2014) and neuroendocrine responses induced by context fear con-ditioning (Yoshida et al. 2014). These experiments thus suggestthat MeA contributes to part of the fear memory engram, butmay not be required for all aspects of fear learning. Further studiesare required to elucidate the precise role of MeA activation follow-ing auditory fear conditioning.

Amygdalostriatal transition areaA population of neurons within the AStr showed learning-specificactivation both in this study and following context fear condi-tioning (Trogrlic et al. 2011). In our previous study, there wasalso some FTL activation in the AStr following exposure to thenovel context, immediate shock, and tone (Trogrlic et al. 2011).However in the present study, there was no FTL activation relatedto context or tone exposure, or recall of fear, and only a small in-crease in the Unpaired condition. This low-FTL expression in thenonlearning controls is most likely due to the extensive habitua-

tion and environmental enrichment prior to training. FTL expres-sion in AStr was therefore predominantly learning specific. Incombination with our context fear conditioning study (Trogrlicet al. 2011) this finding further argues that the FTL+ neurons weidentified in the AStr have a role in fear learning.

The AStr receives extensive projections from LA as well as theaccessory basal nuclei of amygdala, and is likely a major target ofboth of these regions (Jolkkonen et al. 2001). The projection fromthe LA is of particular interest because it raises the possibility thatthe learning-specific neurons we identified in LAvl may projectdirectly to learning-specific neurons in the AStr and form part ofthe fear learning circuit. Using a rat brain slice preparation witha voltage-sensitive imaging system, the electrophysiological char-acteristics of the LA pathways were studied (Wang et al. 2002), andthe LA signal was shown to propagate specifically to AStr andbasolateral nuclei. The LA-AStr signal was characterized as highvelocity, high efficiency and the AStr had the property of temporalsummation of LA signals (Wang et al. 2002). These characteristicsinfer strong functional connection between LA and AStr and sup-port the possibility that this connection forms part of the fearlearning circuit.

The AStr also receives information, via input from cortex andthalamus, from somatosensory, auditory, and visual modalities(LeDoux et al. 1990). The AStr may thus be a stimulus conver-gence region for sensory inputs including footshock, auditory,and contextual stimuli, in addition to receiving inputs from LA.The outputs of AStr are predominantly basal ganglia, includingstriatum, nucleus accumbens, and substantia nigra (Shammah-Lagnado et al. 1999). These structures are involved in movementand higher-order behaviors. The role of AStr in fear learning couldthus involve learning-related signaling to control such fear-related behaviors including avoidance and escape.

Central amygdalaIn an adjacent region to the AStr, within the CeC, there was asmall population of neurons that showed learning-specific activa-tion. These neurons were found only along the lateral border ofthe CeC within a very narrow rostro-caudal extent of �100 mm.They were found at the most rostral extent of the CeC, and alongthe border where it was directly adjacent to the BLA. A number ofother studies have studied c-fos expression in the central amygdala(CeA) following fear conditioning (Pezzone et al. 1992; Beck andFibiger 1995; Milanovic et al. 1998; Radulovic et al. 1998; Dayet al. 2008), but none of these studies determined if any of thec-fos expression was specifically associated with fear learning. Noother studies have identified c-fos+ neurons in this very discretelocation within the CeC.

The CeA has long been associated as an output site of fearfulbehavior (Johansen et al. 2011), but has recently been found to bedirectly involved in the fear-conditioning process (Ciocchi et al.2010; Haubensak et al. 2010; Li et al. 2013). In particular, inhibi-tory neurons in the lateral CeA (CeL, which includes CeC) wereshown to be directly involved in the fear-conditioning process(Ciocchi et al. 2010; Haubensak et al. 2010). Fear learning was as-sociated with modification of excitatory synapses from LA neu-rons projecting onto these inhibitory neurons within CeL (Liet al. 2013).

Whether the highly localized neurons we identified in CeCcorrespond to the learning-specific inhibitory neurons in CeLneeds further investigation. One possibility is that during audito-ry fear conditioning only a small subset of inhibitory neurons inCeL undergoes synaptic modification and correspondingly, onlythese neurons express c-fos. Although most neurons that expressc-fos in amygdala are excitatory (Knapska et al. 2007), it is knownthat some stimuli can stimulate c-fos in inhibitory neurons





(Reznikov et al. 2008). Alternatively, the neurons we identify inCeC may represent a separate population with a different role inthe learning process to the inhibitory neurons described above.The CeC receives nociceptive information from spinal cord viathe parabrachial nucleus and the BLA, and is a possible site of in-tegration of nociceptive and polymodal information in fear con-ditioning (Dong et al. 2010). Indeed fear learning specificallyinduces synaptic potentiation in both parabrachial–CeC andBLA–CeC synapses (Watabe et al. 2013). The learning-specificneurons we identify in CeC may be the post-synaptic neuronsof these potentiated synapses and an integration site for fearconditioning.

Ventromedial hypothalamusFollowing auditory fear conditioning, there was strong learning-specific FTL activation in two discrete regions within the VMH.The largest number of FTL+ neurons was found in the rostralVMH, the same region identified in our context fear conditioningexperiments (Trogrlic et al. 2011). Additionally, we identified apopulation of neurons showing learning-specific FTL activationwithin a caudal region of the VMHvl. We did not detect FTLactivation within VMHvl in the context fear conditioning exper-iments. As discussed above, this may have been due to the gener-ally higher levels of FTL expression in the brains of the mice in thecontext fear conditioning experiments, which is reduced in thecurrent experiments following habituation and environmentalenrichment. FTL expression in both of these regions was veryhighly learning specific with very little expression in the non-learning control mice or following fear memory recall.

The VMH, along with other hypothalamic nuclei, is knownto be involved in regulation of aggression (Kruk et al. 1983; Linet al. 2011) and fearful behavior (Swanson 2000; Martinez et al.2008; Silva et al. 2013). However, the lack of FTL expression inVMH and VMHvl following fear memory recall indicates theVMH is not merely activated by the expression of fear. Experi-ments using predator odor as a US also support involvement ofVMH in fear learning, whereby c-fos expression was more closelycorrelated with fear learning compared with fear expression(Staples et al. 2005). Additionally, there is direct evidence thatthe VMH is involved in conditioned fear, in experiments usingthe fear potentiated startle response. Injections of the GABA ago-nist, muscimol, into the VMH prior to testing for fear potentiatedstartle, produced a reduction in conditioned fear (Santos et al.2008). While this does not establish that VMH neurons are in-volved in fear learning and memory, it distinguishes this regionfrom other hypothalamic nuclei, such as dorsomedial hypothala-mus and premamillary nuclei, which have a clear role in fear be-haviors but no identifiable involvement in conditioned fear(Santos et al. 2008).

The major inputs of the VMH are from basolateral and medialamygdala (Risold et al. 1994; Canteras et al. 2001; Gross andCanteras 2012); where basolateral amygdala provides a link be-tween LA and VMH, medial amygdala shows learning-specific ac-tivation in our experiments. Experiments using antagonists to theneuropeptide Substance P injected into each of these three areasresult in inhibition of fear-potentiated startle (Zhao et al. 2009).Based on this and other data it was proposed that a critical path-way for conditioned fear runs from MeA to VMH, and is regulatedby Substance P. This is consistent with our findings and supportsthe idea that the fear-learning circuit includes a pathway fromMeA to VMH.

Lateral septumAuditory fear conditioning also stimulated FTL expression indorsal LS, an area known to be implicated in processing aversive

stimuli. In this brain region, there was significant FTL activationin Home Cage mice as well as the other nonlearning controlsand the recall group of mice. The degree of learning-specific acti-vation was thus lower than that observed in the other brain re-gions we identified (�60% greater than Unpaired or any othergroup). We did not examine this region in our previous studies(Trogrlic et al. 2011), which were restricted to amygdala andhypothalamus, and thus we did not determine if there was alsolearning-specific neuronal activation following context fearconditioning.

Expression of c-fos has previously been found in LS in a num-ber of different models of fear learning and memory, includingfear conditioning (Pezzone et al. 1992; Beck and Fibiger 1995)and fear potentiated startle (Campeau et al. 1991; Veening et al.2009), however these studies did not analyse if c-fos expressioncorrelated specifically with learning. However, Calandreau et al.(2007) found an increase in the number of c-fos+ neurons in dorsalLS following paired auditory fear conditioning compared with un-paired training, very similar to our results. These findings indicatethat a subpopulation of neurons in dorsal LS express c-fos specifi-cally associated with auditory fear learning.

There is significant evidence that LS is involved in differentemotional states, and in particular in fear and anxiety (Sheehanet al. 2004). There are also a number of studies that have examinedthe role of LS in fear learning and memory. Electrolytic lesions tothe LS result in potentiated freezing following context fear condi-tioning (Sparks and LeDoux 1995), suggesting an involvement ofLS in the conditioning process. However the complete lesioningof LS in these experiments may have simply affected fear expres-sion without affecting fear learning or memory. Experiments in-volving reversible inhibition of LS with lidocaine prior tofear-conditioning training resulted in complete disruption of au-ditory fear conditioning, indicating that LS is required for audito-ry fear learning (Calandreau et al. 2007). In related experimentswith context fear conditioning, reversible inhibition of LS withcobalt chloride prior to fear conditioning had no effect (Reiset al. 2010). These results suggest a specificity of involvement ofLS in auditory, or possibly different forms of cued conditioning,over context conditioning. Further experiments involving gluta-mate agonists and antagonists injected into LS prior to trainingfurther support this idea (Calandreau et al. 2010). In these exper-iments, injection of glutamate agonists into LS promoted audi-tory fear conditioning and disrupted context fear conditioning,whereas the glutamate antagonists had the opposite effect(Calandreau et al. 2010). These findings thus suggest that the LSplays a key role in the specificity of fear conditioning, via selectionof the relevant stimuli.

The LS is therefore implicated as contributing to the selectionof environmental stimuli best able to predict the unconditionedstimulus, and the FTL activation seen in the LS in the currentstudy may be due to this role. Further studies need to be undertak-en to determine if the learning-related c-fos expression is related tosome form of cellular or synaptic plasticity or whether it is associ-ated with some metabolic response in these neurons.

ConclusionsThe regions we identified were all very highly localized and werecomprised of small numbers of activated neurons. A comparisonof the current study on auditory fear conditioning with our pre-vious study with context fear conditioning (Trogrlic et al. 2011)suggests that most of these regions are shared. We identified addi-tional populations in the current study, but this was due to a moreextensive brain screen as well as prior EE and habituation of themice, as discussed above. If these areas are directly involved in





the memory process, this suggests that at least a part of auditoryand context fear learning and memory are contained within smallnumbers of neurons in the same very specific regions of the brain.This implies that the general circuits for auditory and context fearconditioning are shared, at least at the subcortical level. The LS is apossible exception and may not be involved in context fear learn-ing, as discussed above. Additionally, there may be auditory andcontext specificity within these circuits as the auditory-specificand context-specific neurons in these brain regions may be differ-ent, or overlapping, populations.

Each of these populations of neurons may form part of a larg-er fear memory engram, and may be fitted into current hodolog-ical studies. The connections within the amygdala with regardto fear pathways have been extensively described, reciprocallyconnecting both the LA and MeA to the VMH (Canteras et al.1994; LeDoux 2000; Usunoff et al. 2009). AStr is also known toconnect reciprocally with the LA (Jolkkonen et al. 2001), whilethe connections between the VMH and LS have been describedin detail (Risold and Swanson 1996). Each of the brain areasidentified and described in the current study are therefore inter-connected, and these nuclei may exist as nodes in the larger,auditory-fear memory circuit. What is required next is to deter-mine exactly the inputs and output projections of the learn-ing-specific neurons we have identified. Do the FTL+ neuronswe identified project directly or indirectly to each other? This in-formation is required to form an accurate description of the fear-conditioning circuit.

Materials and Methods

AnimalsMale FTL+ mice aged 8–12 wk were obtained from the BiomedicalSciences Animal Facility, University of Melbourne. Mice werehoused in standard 15 cm × 30 cm × 12 cm cages on a 12-hlight–dark cycle, with food and water supplied ad libitum. All ex-periments were conducted with approval of the Animal EthicsCommittee of the University of Melbourne and in accordancewith the guidelines of the National Health and MedicalResearch Council (Australia). At least 2 d prior to the commence-ment of the experiment, mice were singly housed in cages as de-scribed, and relocated to a dedicated behavioral laboratory. Thisfacility consisted of quiet (60 dB) and low light (15–20 Lux) con-ditions on a 12-h light–dark cycle.

Habituation and enrichmentMice were subjected to a slightly modified version of the habi-tuation and environmental enrichment paradigm outlined byNithianantharajah and Murphy 2008. Habituation to the condi-tioning chamber was performed to minimize any association ofthe footshock to context, and consisted of placing the mice intothe conditioning chamber for 3 min each day. As well as habituat-ing the mice to the conditioning chamber they also underwentenvironmental enrichment. This has been previously shown toimprove the specificity of learning outcomes in auditory fear con-ditioning (Nithianantharajah and Murphy 2008). Environmentalenrichment consisted of placing the mice into separate large (53cm × 58 cm × 31 cm) plastic containers containing an assort-ment of objects of varying size, color, and texture (includingcardboard tubes, plastic containers, wooden pet toys, and foampacking material) for 60 min per day. Mice were habituated tothe conditioning chamber and underwent environmental enrich-ment once per day for 21 d prior to auditory fear conditioning.The order in which the mice were exposed to either the condition-ing chamber or the enriched environment was varied each day.Mice then remained in their home cages for 2 d without any han-dling prior to conditioning, to ensure that FTL expression inducedby exposure to the enriched environment and to the conditioningchamber returned to basal levels of FTL expression.

Auditory fear conditioningFear conditioning was conducted using a computerized system(Med Associates, VT, USA). The conditioning chamber was con-tained within a PVC sound-attenuating compartment (16 cm ×14 cm × 13 cm). The floor of the chamber consisted of stainlesssteel rods (diameter 3.2 mm, spaced 5 mm apart), connected toa shocker–scrambler unit capable of delivering electric shocks ofdefined duration and intensity. The chamber was cleaned with70% ethanol between each habituation, training, and testing ses-sions. For auditory fear conditioning, mice were assigned to sepa-rate groups (Context, Tone, Unpaired, Paired, and Recall) andtrained accordingly (see Fig. 1). This ensured that there was a co-hort of mice exposed to each of the different experiential aspectsof the auditory fear conditioning procedure. On the day of train-ing, Paired mice (the learning group) were exposed to a pairedpresentation of the tone and footshock. These animals would un-dergo associative learning, and thus learn to associate the toneand footshock. Paired mice were placed into the chamber for 3min, followed by exposure to the tone (75+5 dB, 10 kHz) for30 sec, which coterminated with a 0.2 mA, 2-sec footshock. Themice then remained within the chamber for a further 30 sec beforebeing returned to their home cages. There were a series of controlgroups in which the mice did not undergo associative learning: (1)unpaired mice were immediately shocked (0.2 mA, 2 sec) upon en-try to the chamber. They then remained in the chamber for 3 min,followed by exposure to the tone (75+5 dB, 10 kHz) for 30 sec.After a further 30 sec in the conditioning chamber, Unpairedmice were returned to their home cage. (2) Tone mice were ex-posed to the chamber for 3 min, followed by exposure to an audi-tory tone (75+5 dB, 10 kHz) for 30 sec. After a further 30 sec inthe conditioning chamber, Tone mice were returned to theirhome cages. (3) Context mice were exposed to the conditioningchamber for 3 min and 30 sec and then returned to their homecages. (4) Recall mice were included to determine the pattern ofFTL induced by the expression of tone-induced fear (freezing).Recall mice had training as for Paired mice but were treated differ-ently for testing (see below).

Paired, Unpaired, Tone, and Context groups of mice re-mained in their home cages for 4 h after training, which allowsfor the detection of FTL activity that has been up-regulated bythe training conditions. Four hours after exposure to the condi-tioning chamber, all mice were tested for context- and tone-asso-ciated fear (Testing), by placing the mice back in the same trainingchamber for 3 min in the absence of the tone, followed by 3 minwith the tone. Freezing behavior was recorded for each 3-min pe-riod, and was defined as the absence of any visible movement ofthe body except for breathing. Moving data were also recordedas the time each mouse spent moving forward laterally to the floorof the training chamber. Immediately following training micewere terminally anesthetized with 100 mL Lethabarb (Virbac) in-jected intraperitoneally. Previous work has shown that the mini-mum time needed to see a significant change in FTL expressiondue to mice experiencing a stimulus such as training or testingfor fear memory is �60 min. As the mice are killed ,10 min afterbeing tested for context- and tone-associated fear, all FTL expres-sion we see in these mice is only due to the training conditions themice experienced 4 h previously.

Recall mice remained in their home cages for 72 h after train-ing, so that FTL expression would return to basal levels. Recallmice were then tested as for the other groups, but returned to theirhome cages for 4 h before being anesthetized. Any changes in FTLexpression in these mice is due to the recall of fear and expressinga fear-conditioned response, such as freezing.

Home cage controls were also included, and consisted ofmice taken directly from their home cage with no other treatmentprior to anesthetization.

Histochemical proceduresImmediately following anesthesia, mice were transcardially per-fused with 12 mL 10% sucrose, 2.5 × 10–3 units per microliterHeparin (Sigma-Aldrich, Australia) in analytical grade water, fol-lowed by 24 mL of 4% paraformaldehyde (PFA, Merck), and





0.005% gluteraldehyde (EM sciences) in 0.1 M phosphate buffer(pH 7.4). After 30 min, the brains were dissected out and post-fixed in 4% PFA, 0.1 M phosphate buffer for 30 min, rinsed twicewith phosphate-buffered saline (PBS), and submerged in cryopro-tectant (20% sucrose in 0.1 M PBS) for 48 h. Brains were then snap-frozen in OCT (Sakura Finetek) and kept at 280˚C. Coronal sec-tions (50 mm) were cut on a cryostat and transferred into a24-well tissue culture plate containing PBS, with each well con-taining 2–3 sections.

The PBS was aspirated from the wells, and replaced with 400mL per well of assay buffer (10 mM potassium ferricyanide, 10 mMpotassium ferrocyanide, 40 mM MgCl2, and 2 mg/mL 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal; Roche) in 0.1 MPBS) for 24 h at room temperature, with agitation. Volumeswere kept the same for all experiments in order to standardizethe conditions of the enzymatic reaction. The sections werethen rinsed with PBS and stored in 4% PFA until mounting in0.5% gelatin solution onto glass slides coated with 0.5% gelatin,10% potassium chromium alum. Sections were left to dry, dehy-drated in histolene and coverslipped (Murphy et al. 2007).

FTL analysisStained sections were screened microscopically (Olympus BX61)and brain regions were identified by comparison with a mousebrain atlas (Franklin and Paxinos 2008). Initially, regions oflearning-specific FTL expression throughout the brain wereidentified qualitatively by comparison of sections from Pairedmice brains with matching sections from Unpaired brains.Subsequently, slides were scanned using a MIRAX SCAN (Zeiss,Germany) and studied using MIRAX viewer software, enablingside by side comparisons of each section from Paired andUnpaired mouse brains. Sections from all areas of the brain frombregma 3.20 mm to bregma 28.00 mm (excluding cerebellum,which had very little FTL expression) were examined and system-atically compared (Ali et al. 2009) this way to identify any addi-tional learning-specific brain regions. Areas that showed aqualitative difference between the Paired and Unpaired condi-tions in levels of activation were then examined quantitativelyin the corresponding sections from all groups. The counting wasperformed in defined regions of interest (ROI) using a frame gen-erated by the ROI tool of analySIS software (Olympus) of the sameshape and size for each brain region (see Results for further detailsof which areas were counted).

FTL cell counts were done using an Olympus microscope(BX61) at 100× magnification. For all areas where neurons werecounted, the area of section was examined in detail by focusingthroughout its thickness. In the LAvl only neurons with the fol-lowing characteristics were counted; neurons had to be within100 mm of the border of the LaVL with the external capsule,were FTL+ through the entire cell body, and had a stellate mor-phology with at least two processes. For MeA, AStr, and CeC re-gions, all cells that were clearly identifiable were counted,even cells with relatively weak FTL staining. In the VMH, onlythe large stained neurons with visible processes within the definedregion (see Results) were included in the count. For the LS region,FTL+ neurons were counted within a 400 mm × 600 mm ovaleither side of the midline throughout the entire LS (bregma1.10–0.14 mm), and the three consecutive sections with the high-est counts were pooled for each brain. For quantification of eachregion, the following numbers of mice were used: Home Cagen ¼ 7, Context n ¼ 6, Tone n ¼ 8, Unpaired n ¼ 9, Paired n ¼ 13,Recall n ¼ 5.

To determine the fraction of neurons activated in each re-gion, the density of neurons was estimated by counting the num-ber of Nissl+ cells at least 10 mm in diameter in Nissl-stainedsections of known thickness for each counting region from amouse brain atlas (Franklin and Paxinos 2008). Each density wasthen multiplied by the volume of the corresponding countingregion. Total neuron numbers for each counting region were asfollows: LAvl border region 1560; AStr 860; MeApd 5850; CeC910; VMH 3000; VMHvl 740; LSI 1590. Numbers of activated neu-rons were represented as a percentage of this cell number.

Statistical significance was determined using a one-wayANOVA and Bonferroni’s multiple comparison test comparingHome cage to Context, Paired to Unpaired, and Paired to Recall.Prism software (GraphPad Software) was used for statisticalanalysis.

AcknowledgmentsWe thank Rowena Mortimer for technical assistance and MayaKesar and Sarah Travener for mouse husbandry.

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Received November 25, 2014; accepted in revised form April 28, 2015.





10.1101/lm.037663.114Access the most recent version at doi: 22:2015, Learn. Mem.

Christopher W. Butler, Yvette M. Wilson, Jenny M. Gunnersen, et al. activated by auditory fear conditioningwithin amygdala, hypothalamus, and lateral septum are specifically Tracking the fear memory engram: discrete populations of neurons

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